1. Technical Field
The present invention is related to a fluorometric assay apparatus and a fluorometric assay method, and in particular to a fluorometric assay apparatus and fluorometric assay method employing as a light source plural types of LED for generating ultraviolet radiation and visible light.
2. Related Art
In fields of biochemistry, for example as described in Japanese Patent Laid-Open No. 2005-283322, imaging devices are proposed that capture images of subjects such as fluorescent light-emitting specimens marked with fluorescent stains that emit fluorescent light on being illuminated with excitation light or chemical light-emitting specimens that have been placed in contact with a chemical light-emitting substrate and emit light.
Products for such fields are available with highly monochromatic near infrared, red, green, blue, ultraviolet, and white light sources. Such products can cause light of each desired color to be emitted under control from computer software, and are capable of mechanically switching between each light source.
In such cases, for example with fluorescent stains EtBr (ethidium bromide) and SYBR Green (SYBR is a registered trade mark of Molecular Probe Inc., same is applied hereinafter), when both are respectively appropriately excited and detected, an excitation light source appropriate to the absorption characteristics of each stain is respectively selected and employed. This means that there is a requirement to appropriately select light sources such as an ultraviolet light source for EtBr and a blue light source for SYBR Green.
Namely, generally a light source unit is built with a light source and a first filter (excitation filter) placed directly after the light source. However an imaging technician needs to perform operations to switch over light source units in order to use a light source and first filter with spectral characteristics optimized to the specific fluorescent stain that was selected by the imaging technician. A second filter (detection filter) is further disposed immediately in front of an image pick-up element, and the excitation wavelength component is cut out so that excitation light is not incident on the image pick-up element.
As detection apparatuses capable of handling both EtBr and SYBR Green (for example Bio-Rad ChemiDoc) employing an ultraviolet fluorescent lamp that emits a main light band of wavelengths capable of exciting both EtBr and SYBR Green are known.
As described in for example JP-A No. 2008-145405, configurations exist that simultaneously illuminate excitation light of two different wavelengths as an excitation light source.
As described in for example JP-A No. 2005-172614, configurations exist that include LEDs that emit blue light and LEDs that emit ultraviolet light arrayed in a staggered grid pattern as a light source.
As described in JP-A No. 2009-300356, configurations exist in which two excitation light sources are caused to be illuminated intermittently at different frequencies, and in which corresponding fluorescent light is detected.
As described in JP-A No. 2010-091456, configurations exist that include LED light sources disposed in a regular pattern on a substrate configured such that light intensity can be controlled independently.
As described in JP-A No. 2001-083090, configurations exist that include, as excitation light sources, plural LED light sources disposed in a regular pattern on a substrate, configured such that the light intensity and the light emission wavelength of the excitation light from the plural LED light sources can be controlled independently.
However, in systems using an ultraviolet fluorescent light lamp that emits a main light band of wavelengths capable of exciting both EtBr and SYBR Green, the excitation efficiency with respect to the fluorescent stain SYBR Green is far from high, and as well as the shortcoming of heat generated by the light source affecting the specimen, there are also disadvantages with respect to compact design.
In the configuration of JP-A No. 2008-145405, since the way the excitation light hits specimens varies depending on the attachment positions of each of the light sources, it is not possible for each wavelength to illuminate equally, and effort is also required to prepare appropriate excitation filters for each light source and to switch them over as required.
In the configuration of JP-A No. 2005-172614, it is necessary to switch over detection filters on the detector side in order to obtain images for each wavelength, and also, as this configuration does not specifically employ fluorescent stains, there is no concept of cutting out or passing excitation light using filters.
In the configuration of JP-A No. 2009-300356, a high cost dichroic filter (interference filter) is used as an excitation light filter in order to illuminate a specimen with two excitation light sources along the same axis, and it is not possible to use a single excitation light filter for plural excitation light sources.
In the configuration of JP-A No. 2010-091456, it is necessary to prepare fluorescent light filters for each fluorescent stain in order for fluorescent light of different wavelength bands depending on each fluorescent stain to be transmitted. As well as an increase in the number and types of filter, there is also the shortcoming of the effort for switching over filters.
In the configuration of JP-A No. 2001-083090, configuration is for a light source for a microtiter plate furnished with plural hollows in for example a glass plate, with one LED employed per hollow (well), such that plural filters need to be provided in order to filter excitation light independently of each other. This also has the disadvantage of an increase in the number and types of filter, and the effort for switching over filters.
Operations to switch the light source unit over according to the fluorescent stain used, as described above, force an unnecessary burden on an operator (imaging technician).